Over the last 56 years, researchers have been developing methods and technologies to assist in the determination of nucleic acid sequences in biological samples. The ability to sequence DNA and RNA accurately has had a great impact in numerous research fields. The sequencing of the human genome was completed in 2003, after 13 years of international collaboration and investment of USD 3 billion. The Human Genome Project used Sanger sequencing, the principal method of DNA sequencing since its invention in the 1970s. Today, the demand for sequencing is growing exponentially, with large amounts of genomic DNA needing to be analyzed quickly, cheaply, and accurately. Thanks to new sequencing technologies known collectively as Next Generation Sequencing.

Next generation sequencing (NGS), also known as high throughput sequencing or second generation sequencing or short read sequencing, is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA.

The main difference between Sanger sequencing and NGS stems from sequencing volume, with NGS allowing the processing of millions of reactions in parallel, resulting in high-throughput, higher sensitivity, speed and reduced cost. A plethora of genome sequencing projects that took many years with Sanger sequencing methods could now be completed within hours using NGS.

There are several main steps that must be tailored to the target (RNA or DNA) and sequencing system selected. The main steps of NGS includes sample preparation (pre-processing), library preparation, PCR amplification, sequencing and data analysis (Post-processing).

Library preparation includes DNA fragmentation either enzymatically or by sonication, end repairing and adaptor ligation.

Library amplification is required so that the received signal from the sequencer is strong enough to be detected accurately. The two most common PCR amplification methods are emulsion PCR and bridge PCR.

Several competing methods of NGS have been developed by different companies including 454 Pyrosequencing, sequencing by ligation (SOLiD), ion torrent semiconductor sequencing and reversible terminator sequencing (illumina).

NGS can generate two types of reads i.e, single end reads and paired end reads, depending on method of choice.

NGS has enabled researchers to collect vast quantities of genomic sequencing data. This technology has a plethora of applications, such as: outbreak Management, diagnosing and understanding complex diseases, whole-genome sequencing, transcriptome sequencing, cancer treatments, detection of viruses, surveillance of antimicrobial resistance and many more.

Now, third (3G) and fourth (4G) generation technologies have been evolved that work on different underlying principles.

This course is a valuable resource for students and researchers related to molecular biology, forensic science, medical laboratory technology, biotechnology, and genetics.

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